Whether amyloid formation in stress conditions serves any beneficial purpose or is instead simply a pathologic outcome of the stress is an important open question. Elucidating the cellular triggers and physiological consequences of amyloid formation therefore represents a critical barrier to advancing our understanding of amyloid biology. The applicant's long-term goal is to use the yeast model system to understand the stimuli that trigger amyloid formation, the mechanisms underlying this response and its physiological significance. The objective of this proposal is to decipher the ribosome-associated mechanisms that regulate prion formation for three yeast prions and the impact of one of those prions on the yeast proteome. The central hypothesis is that ribosome-associated factors, such as the Ribosome-Associated Complex (RAC), play a central role in regulating prion formation in response to environmental stimuli and that the resulting prion phenotypes have important physiological implications. The hypothesis is based on the applicant's published work and preliminary data. The rationale for the proposed research is that deciphering the contributions of ribosome-associated processes in prion formation is a critical step in understanding the overall regulation and physiological impacts of amyloid formation in order to facilitate pharmacological manipulation. Using the yeast prions [PSI+], [URE3] or [RNQ+] as models this hypothesis will be tested by pursuing three specific aims: 1) Determine the extent to which co- translational prion formation occurs under stress and non-stress conditions; 2) Identify ribosome-associated mechanisms that regulate prion formation; and 3) Determine the impact of prion formation on the yeast proteome. Genetic, biochemical and cell biological approaches, which have been established as feasible in the applicant's hands, will be employed to accomplish these goals. The proposed work is innovative because it represents a substantial departure from the status quo by examining the earliest possible time-point in prionogenesis - during synthesis of the prion protein - and by assessing the physiological consequences of prion switching. Additionally, these experiments will be enriched by the applicant's development of an innovative new prion reporter system that enables rapid, quantitative measurements of prion formation and prion variant strength in individual cells in real time. The contribution of the proposed research s expected to be the elucidation of ribosome-associated mechanisms regulating co-translational amyloid formation in various environmental conditions and the resulting consequences on cellular physiology. This contribution will be significant because co-translational amyloid formation constitutes the earliest misfolding event against which pharmacological intervention could be targeted; thus, an understanding of the underlying triggers and regulatory mechanisms of co-translational amyloidogenesis is urgently needed to advance the field and could provide a basis for targeting mammalian homologs in pharmacological interventions of mammalian protein misfolding disorders.